A remarkable feature of granulopoiesis is the regulated production and release of neutrophils (PMN) to maintain homeostatic levels in the circulation. The granulocyte colony-stimulating factor receptor (G-CSFR) plays a critical role in this process as the major regulator of myeloid cell proliferation and differentiation and as a modulator of PMN and stem cell mobilization. Deregulation in G-CSFR expression and signaling is linked to neutrophil deficiencies and human leukemias. Despite the critical role that the G-CSFR plays in regulating granulopoiesis and the current widespread clinical use of G-CSF for prevention and/or treatment of neutropenia and for stem cell mobilization, the molecular mechanisms that downregulate G-CSFR expression and signaling to prevent uncontrolled myeloid cell expansion remain unknown. The overall goal of this proposal is to determine the role of ubiquitination in inactivation of the granulocyte colony-stimulating factor receptor (G-CSFR). We hypothesize that ubiquitin-modification of the G-CSFR regulates its expression at the cell surface and its intracellular fate and signaling potency and that defects in G-CSFR ubiquitination contribute to human disease. We will test our hypothesis in the studies proposed in this application using ultrastructural analyses as well as molecular, biochemical, and functional approaches to analyze the dynamics of G-CSFR ubiquitination on receptor trafficking and signaling. We will also investigate whether forced ubiquitination of internalization-incompetent mutant G-CSFR forms from patients with acute myelogenous leukemia (AML) corrects aberrant receptor surface expression and trafficking to reverse the hyperproliferative phenotype. Retroviral transduction of G-CSFR variants into primary cells from G-CSFR null (-/-) mice will allow us to confirm results obtained in cell lines and establish their physiological relevance. Determination of the functional consequences of G-CSFR ubiquitination will provide new insights into the fundamental mechanisms of G-CSFR desensitization which may have therapeutic application for modulation of granulopoiesis and progenitor cell mobilization.